Effects of small molecule-induced dimerization on the programmed death ligand 1 protein life cycle.

The programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) checkpoint blockade is central to Immuno-Oncology based therapies, and alternatives to antibody blockers of this interaction are an active area of research due to antibody related toxicities. Recently, small molecule compounds that induce PD-L1 dimerization and occlusion of PD-1 binding site have been identified and developed for clinical trials. This mechanism invokes an oligomeric state of PD-L1 not observed in cells previously, as PD-L1 is generally believed to function as a monomer. Therefore, understanding the cellular lifecycle of the induced PD-L1 dimer is of keen interest. Our report describes a moderate but consistent increase in the PD-L1 rate of degradation observed upon protein dimerization as compared to the monomer counterpart. This subtle change, while not resolved by measuring total PD-L1 cellular levels by western blotting, triggered investigations of the overall protein distribution across various cellular compartments. We show that PD-L1 dimerization does not lead to rapid internalization of neither transfected nor endogenously expressed protein forms. Instead, evidence is presented that dimerization results in retention of PD-L1 intracellularly, which concomitantly correlates with its reduction on the cell surface. Therefore, the obtained data for the first time points to the ability of small molecules to induce dimerization of the newly synthesized PD-L1 in addition to the protein already present on the plasma membrane. Overall, this work serves to improve our understanding of this important target on a molecular level in order to guide advances in drug development.

Hepatitis B Virus X Protein Function Requires Zinc Binding.

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined. In this study, we performed biophysical analyses of recombinant HBx and functional analysis of HBx mutants in HBV-infected primary human hepatocytes (PHH) to identify key regions and residues that are required for HBx function. We determined that recombinant HBx is soluble and exhibits stoichiometric zinc binding when expressed in the presence of DDB1. Mass spectrometry-based hydrogen-deuterium exchange and cysteine-specific chemical footprinting of the HBx:DDB1 complex identified several HBx cysteine residues (located between amino acids 61 and 137) that are likely involved in zinc binding. These cysteine residues did not form disulfide bonds in HBx expressed in human cells. In line with the biophysical data, functional analysis demonstrated that HBx amino acids 45 to 140 are required for Smc6 degradation and HBV transcription in PHH. Furthermore, site-directed mutagenesis determined that C61, C69, C137, and H139 are necessary for HBx function, although they are likely not essential for DDB1 binding. This CCCH motif is highly conserved in HBV as well as in the X proteins from various mammalian hepadnaviruses. Collectively, our data indicate that the essential HBx cysteine and histidine residues form a zinc-binding motif that is required for HBx function.IMPORTANCE The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses HBV transcription. HBV counters this restriction by expressing HBV X protein (HBx), which redirects a host ubiquitin ligase to target Smc5/6 for degradation. Despite this recent advance in understanding HBx function, the key regions and residues of HBx required for Smc5/6 degradation have not been determined. In the present study, we performed biochemical, biophysical, and cell-based analyses of HBx. By doing so, we mapped the minimal functional region of HBx and identified a highly conserved CCCH motif in HBx that is likely responsible for coordinating zinc and is essential for HBx function. We also developed a method to produce soluble recombinant HBx protein that likely adopts a physiologically relevant conformation. Collectively, this study provides new insights into the HBx structure-function relationship and suggests a new approach for structural studies of this enigmatic viral regulatory protein.

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody. In this study, we developed a novel monoclonal antibody that enables detection of HBx protein in HBV-infected primary human hepatocytes (PHH) by Western blotting and immunofluorescence. Confocal imaging studies with this antibody demonstrated that HBx is predominantly located in the nucleus of HBV-infected PHH, where it exhibits a diffuse staining pattern. In contrast, a DDB1-binding-deficient HBx mutant was detected in both the cytoplasm and nucleus, suggesting that the DDB1 interaction plays an important role in the nuclear localization of HBx. Our study also revealed that HBx is expressed early after infection and has a short half-life (∼3 h) in HBV-infected PHH. In addition, we found that treatment with small interfering RNAs (siRNAs) that target DDB1 or HBx mRNA decreased HBx protein levels and led to the reappearance of Smc6 in the nuclei of HBV-infected PHH. Collectively, these studies provide the first spatiotemporal analysis of HBx in a natural infection system and also suggest that HBV transcriptional silencing by Smc5/6 can be restored by therapeutic targeting of HBx.IMPORTANCE Hepatitis B virus X protein (HBx) is a promising drug target since it promotes the degradation of the host structural maintenance of chromosomes 5/6 complex (Smc5/6) that inhibits HBV transcription. To date, it has not been possible to study HBx in physiologically relevant cell culture systems due to the lack of a highly specific and selective HBx antibody. In this study, we developed a novel monoclonal HBx antibody and performed a spatiotemporal analysis of HBx in a natural infection system. This revealed that HBx localizes to the nucleus of infected cells, is expressed shortly after infection, and has a short half-life. In addition, we demonstrated that inhibiting HBx expression or function promotes the reappearance of Smc6 in the nucleus of infected cells. These data provide new insights into HBx and underscore its potential as a novel target for the treatment of chronic HBV infection.

The KN-93 Molecule Inhibits Calcium/Calmodulin-Dependent Protein Kinase II (CaMKII) Activity by Binding to Ca2+/CaM.

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that transmits calcium signals in various cellular processes. CaMKII is activated by calcium-bound calmodulin (Ca2+/CaM) through a direct binding mechanism involving a regulatory C-terminal α-helix in CaMKII. The Ca2+/CaM binding triggers transphosphorylation of critical threonine residues proximal to the CaM-binding site leading to the autoactivated state of CaMKII. The demonstration of its critical roles in pathophysiological processes has elevated CaMKII to a key target in the management of numerous diseases. The molecule KN-93 is the most widely used inhibitor for studying the cellular and in vivo functions of CaMKII. It is widely believed that KN-93 binds directly to CaMKII, thus preventing kinase activation by competing with Ca2+/CaM. Herein, we employed surface plasmon resonance, NMR, and isothermal titration calorimetry to characterize this presumed interaction. Our results revealed that KN-93 binds directly to Ca2+/CaM and not to CaMKII. This binding would disrupt the ability of Ca2+/CaM to interact with CaMKII, effectively inhibiting CaMKII activation. Our findings also indicated that KN-93 can specifically compete with a CaMKIIδ-derived peptide for binding to Ca2+/CaM. As indicated by the surface plasmon resonance and isothermal titration calorimetry data, apparently at least two KN-93 molecules can bind to Ca2+/CaM. Our findings provide new insight into how in vitro and in vivo data obtained with KN-93 should be interpreted. They further suggest that other Ca2+/CaM-dependent, non-CaMKII activities should be considered in KN-93-based mechanism-of-action studies and drug discovery efforts.

Biochemical characterization and structure determination of a potent, selective antibody inhibitor of human MMP9.

Matrix metalloproteinase 9 (MMP9) is a member of a large family of proteases that are secreted as inactive zymogens. It is a key regulator of the extracellular matrix, involved in the degradation of various extracellular matrix proteins. MMP9 plays a pathological role in a variety of inflammatory and oncology disorders and has long been considered an attractive therapeutic target. GS-5745, a potent, highly selective humanized monoclonal antibody inhibitor of MMP9, has shown promise in treating ulcerative colitis and gastric cancer. Here we describe the crystal structure of GS-5745·MMP9 complex and biochemical studies to elucidate the mechanism of inhibition of MMP9 by GS-5745. GS-5745 binds MMP9 distal to the active site, near the junction between the prodomain and catalytic domain, and inhibits MMP9 by two mechanisms. Binding to pro-MMP9 prevents MMP9 activation, whereas binding to active MMP9 allosterically inhibits activity.

Direct binding of ledipasvir to HCV NS5A: mechanism of resistance to an HCV antiviral agent.

Ledipasvir, a direct acting antiviral agent (DAA) targeting the Hepatitis C Virus NS5A protein, exhibits picomolar activity in replicon cells. While its mechanism of action is unclear, mutations that confer resistance to ledipasvir in HCV replicon cells are located in NS5A, suggesting that NS5A is the direct target of ledipasvir. To date co-precipitation and cross-linking experiments in replicon or NS5A transfected cells have not conclusively shown a direct, specific interaction between NS5A and ledipasvir. Using recombinant, full length NS5A, we show that ledipasvir binds directly, with high affinity and specificity, to NS5A. Ledipasvir binding to recombinant NS5A is saturable with a dissociation constant in the low nanomolar range. A mutant form of NS5A (Y93H) that confers resistance to ledipasvir shows diminished binding to ledipasvir. The current study shows that ledipasvir inhibits NS5A through direct binding and that resistance to ledipasvir is the result of a reduction in binding affinity to NS5A mutants.

The N-Terminal Domain of SIRT1 Is a Positive Regulator of Endogenous SIRT1-Dependent Deacetylation and Transcriptional Outputs.

The NAD+-dependent protein deacetylase SIRT1 regulates energy metabolism, responses to stress, and aging by deacetylating many different proteins, including histones and transcription factors. The mechanisms controlling SIRT1 enzymatic activity are complex and incompletely characterized, yet essential for understanding how to develop therapeutics that target SIRT1. Here, we demonstrate that the N-terminal domain of SIRT1 (NTERM) can trans-activate deacetylation activity by physically interacting with endogenous SIRT1 and promoting its association with the deacetylation substrate NF-κB p65. Two motifs within the NTERM domain contribute to activation of SIRT1-dependent activities, and expression of one of these motifs in mice is sufficient to lower fasting glucose levels and improve glucose tolerance in a manner similar to overexpression of SIRT1. Our results provide insights into the regulation of SIRT1 activity and a rationale for pharmacological control of SIRT1-dependent activities.

Identification of luminal Loop 1 of Scap protein as the sterol sensor that maintains cholesterol homeostasis.

Cellular cholesterol homeostasis is maintained by Scap, an endoplasmic reticulum (ER) protein with eight transmembrane helices. In cholesterol-depleted cells, Scap transports sterol regulatory element-binding proteins (SREBPs) to the Golgi, where the active fragment of SREBP is liberated by proteases so that it can activate genes for cholesterol synthesis. When ER cholesterol increases, Scap binds cholesterol, and this changes the conformation of cytosolic Loop 6, which contains the binding site for COPII proteins. The altered conformation precludes COPII binding, abrogating movement to the Golgi. Consequently, cholesterol synthesis declines. Here, we identify the cholesterol-binding site on Scap as Loop 1, a 245-amino acid sequence that projects into the ER lumen. Recombinant Loop 1 binds sterols with a specificity identical to that of the entire Scap membrane domain. When tyrosine 234 in Loop 1 is mutated to alanine, Loop 6 assumes the cholesterol-bound conformation, even in sterol-depleted cells. As a result, full-length Scap(Y234A) cannot mediate SREBP processing in transfected cells. These results indicate that luminal Loop 1 of Scap controls the conformation of cytosolic Loop 6, thereby determining whether cells produce cholesterol.

The structure of the NPC1L1 N-terminal domain in a closed conformation.

BACKGROUND: NPC1L1 is the molecular target of the cholesterol lowering drug Ezetimibe and mediates the intestinal absorption of cholesterol. Inhibition or deletion of NPC1L1 reduces intestinal cholesterol absorption, resulting in reduction of plasma cholesterol levels. PRINCIPAL FINDINGS: Here we present the 2.8 Å crystal structure of the N-terminal domain (NTD) of NPC1L1 in the absence of cholesterol. The structure, combined with biochemical data, reveals the mechanism of cholesterol selectivity of NPC1L1. Comparison to the cholesterol free and bound structures of NPC1(NTD) reveals that NPC1L1(NTD) is in a closed conformation and the sterol binding pocket is occluded from solvent. CONCLUSION: The structure of NPC1L1(NTD) reveals a degree of flexibility surrounding the entrance to the sterol binding pocket, suggesting a gating mechanism that relies on multiple movements around the entrance to the sterol binding pocket.

Crystal structure of Spot 14, a modulator of fatty acid synthesis.

Spot 14 (S14) is a protein that is abundantly expressed in lipogenic tissues and is regulated in a manner similar to other enzymes involved in fatty acid synthesis. Deletion of S14 in mice decreased lipid synthesis in lactating mammary tissue, but the mechanism of S14's action is unknown. Here we present the crystal structure of S14 to 2.65 Å and biochemical data showing that S14 can form heterodimers with MIG12. MIG12 modulates fatty acid synthesis by inducing the polymerization and activity of acetyl-CoA carboxylase, the first committed enzymatic reaction in the fatty acid synthesis pathway. Coexpression of S14 and MIG12 leads to heterodimers and reduced acetyl-CoA carboxylase polymerization and activity. The structure of S14 suggests a mechanism whereby heterodimer formation with MIG12 attenuates the ability of MIG12 to activate ACC.

Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes.

Water-soluble Niemann-Pick C2 (NPC2) and membrane-bound NPC1 are cholesterol-binding lysosomal proteins required for export of lipoprotein-derived cholesterol from lysosomes. The binding site in NPC1 is located in its N-terminal domain (NTD), which projects into the lysosomal lumen. Here we perform alanine-scanning mutagenesis to identify residues in NPC2 that are essential for transfer of cholesterol to NPC1(NTD). Transfer requires three residues that form a patch on the surface of NPC2. We previously identified a patch of residues on the surface of NPC1(NTD) that are required for transfer. We present a model in which these two surface patches on NPC2 and NPC1(NTD) interact, thereby opening an entry pore on NPC1(NTD) and allowing cholesterol to transfer without passing through the water phase. We refer to this transfer as a hydrophobic handoff and hypothesize that this handoff is essential for cholesterol export from lysosomes.

Induced polymerization of mammalian acetyl-CoA carboxylase by MIG12 provides a tertiary level of regulation of fatty acid synthesis.

Acetyl-CoA carboxylase (ACC), the first committed enzyme in fatty acid (FA) synthesis, is regulated by phosphorylation/dephosphorylation, transcription, and an unusual mechanism of protein polymerization. Polymerization of ACC increases enzymatic activity and is induced in vitro by supraphysiological concentrations of citrate (> 5 mM). Here, we show that MIG12, a 22 kDa cytosolic protein of previously unknown function, binds to ACC and lowers the threshold for citrate activation into the physiological range (< 1 mM). In vitro, recombinant MIG12 induced polymerization of ACC (as determined by nondenaturing gels, FPLC, and electron microscopy) and increased ACC activity by > 50-fold in the presence of 1 mM citrate. In vivo, overexpression of MIG12 in liver induced ACC polymerization, increased FA synthesis, and produced triglyceride accumulation and fatty liver. Thus, in addition to its regulation by phosphorylation and transcription, ACC is regulated at a tertiary level by MIG12, which facilitates ACC polymerization and enhances enzymatic activity.

Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol.

LDL delivers cholesterol to lysosomes by receptor-mediated endocytosis. Exit of cholesterol from lysosomes requires two proteins, membrane-bound Niemann-Pick C1 (NPC1) and soluble NPC2. NPC2 binds cholesterol with its isooctyl side chain buried and its 3beta-hydroxyl exposed. Here, we describe high-resolution structures of the N-terminal domain (NTD) of NPC1 and complexes with cholesterol and 25-hydroxycholesterol. NPC1(NTD) binds cholesterol in an orientation opposite to NPC2: 3beta-hydroxyl buried and isooctyl side chain exposed. Cholesterol transfer from NPC2 to NPC1(NTD) requires reorientation of a helical subdomain in NPC1(NTD), enlarging the opening for cholesterol entry. NPC1 with point mutations in this subdomain (distinct from the binding subdomain) cannot accept cholesterol from NPC2 and cannot restore cholesterol exit from lysosomes in NPC1-deficient cells. We propose a working model wherein after lysosomal hydrolysis of LDL-cholesteryl esters, cholesterol binds NPC2, which transfers it to NPC1(NTD), reversing its orientation and allowing insertion of its isooctyl side chain into the outer lysosomal membranes.

Antagonism of secreted PCSK9 increases low density lipoprotein receptor expression in HepG2 cells.

PCSK9 is a secreted protein that degrades low density lipoprotein receptors (LDLRs) in liver by binding to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. It is not known whether PCSK9 causes degradation of LDLRs within the secretory pathway or following secretion and reuptake via endocytosis. Here we show that a mutation in the LDLR EGF-A domain associated with familial hypercholesterolemia, H306Y, results in increased sensitivity to exogenous PCSK9-mediated cellular degradation because of enhanced PCSK9 binding affinity. The crystal structure of the PCSK9-EGF-A(H306Y) complex shows that Tyr-306 forms a hydrogen bond with Asp-374 in PCSK9 at neutral pH, which strengthens the interaction with PCSK9. To block secreted PCSK9 activity, LDLR (H306Y) subfragments were added to the medium of HepG2 cells stably overexpressing wild-type PCSK9 or gain-of-function PCSK9 mutants associated with hypercholesterolemia (D374Y or S127R). These subfragments blocked secreted PCSK9 binding to cell surface LDLRs and resulted in the recovery of LDLR levels to those of control cells. We conclude that PCSK9 acts primarily as a secreted factor to cause LDLR degradation. These studies support the concept that pharmacological inhibition of the PCSK9-LDLR interaction extracellularly will increase hepatic LDLR expression and lower plasma low density lipoprotein levels.

NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes.

Egress of lipoprotein-derived cholesterol from lysosomes requires two lysosomal proteins, polytopic membrane-bound Niemann-Pick C1 (NPC1) and soluble Niemann-Pick C2 (NPC2). The reason for this dual requirement is unknown. Previously, we showed that the soluble luminal N-terminal domain (NTD) of NPC1 (amino acids 25-264) binds cholesterol. This NTD is designated NPC1(NTD). We and others showed that soluble NPC2 also binds cholesterol. Here, we establish an in vitro assay to measure transfer of [(3)H]cholesterol between these two proteins and phosphatidylcholine liposomes. Whereas NPC2 rapidly donates or accepts cholesterol from liposomes, NPC1(NTD) acts much more slowly. Bidirectional transfer of cholesterol between NPC1(NTD) and liposomes is accelerated >100-fold by NPC2. A naturally occurring human mutant of NPC2 (Pro120Ser) fails to bind cholesterol and fails to stimulate cholesterol transfer from NPC1(NTD) to liposomes. NPC2 may be essential to deliver or remove cholesterol from NPC1, an interaction that links both proteins to the cholesterol egress process from lysosomes. These findings may explain how mutations in either protein can produce a similar clinical phenotype.

Molecular basis for LDL receptor recognition by PCSK9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) posttranslationally regulates hepatic low-density lipoprotein receptors (LDLRs) by binding to LDLRs on the cell surface, leading to their degradation. The binding site of PCSK9 has been localized to the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR. Here, we describe the crystal structure of a complex between PCSK9 and the EGF-A domain of the LDLR. The binding site for the LDLR EGF-A domain resides on the surface of PCSK9's subtilisin-like catalytic domain containing Asp-374, a residue for which a gain-of-function mutation (Asp-374-Tyr) increases the affinity of PCSK9 toward LDLR and increases plasma LDL-cholesterol (LDL-C) levels in humans. The binding surface on PCSK9 is distant from its catalytic site, and the EGF-A domain makes no contact with either the C-terminal domain or the prodomain. Point mutations in PCSK9 that altered key residues contributing to EGF-A binding (Arg-194 and Phe-379) greatly diminished binding to the LDLR's extracellular domain. The structure of PCSK9 in complex with the LDLR EGF-A domain defines potential therapeutic target sites for blocking agents that could interfere with this interaction in vivo, thereby increasing LDLR function and reducing plasma LDL-C levels.

Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: oxysterols block transport by binding to Insig.

Cholesterol synthesis in animals is controlled by the regulated transport of sterol regulatory element-binding proteins (SREBPs) from the endoplasmic reticulum to the Golgi, where the transcription factors are processed proteolytically to release active fragments. Transport is inhibited by either cholesterol or oxysterols, blocking cholesterol synthesis. Cholesterol acts by binding to the SREBP-escort protein Scap, thereby causing Scap to bind to anchor proteins called Insigs. Here, we show that oxysterols act by binding to Insigs, causing Insigs to bind to Scap. Mutational analysis of the six transmembrane helices of Insigs reveals that the third and fourth are important for Insig's binding to oxysterols and to Scap. These studies define Insigs as oxysterol-binding proteins, explaining the long-known ability of oxysterols to inhibit cholesterol synthesis in animal cells.

Direct binding of cholesterol to the purified membrane region of SCAP: mechanism for a sterol-sensing domain.

Mammalian cells control their membrane composition by regulating the vesicular transport of membrane-bound sterol regulatory element binding proteins (SREBPs) from endoplasmic reticulum (ER) to Golgi. Transport is blocked by cholesterol, which triggers SCAP, the SREBP escort protein, to bind to Insigs, which are ER retention proteins. The cholesterol trigger mechanism is unknown. Using recombinant SCAP purified in detergent, we show that cholesterol acts by binding with high affinity and specificity to the 767 amino acid octahelical membrane region of SCAP. This octahelical region contains a conserved pentahelical sterol-sensing domain found in six other polytopic membrane proteins. We show that the membrane domain of SCAP is a tetramer and that cholesterol binding is inhibited by cationic amphiphiles, raising the possibility of allosteric regulation by positively charged phospholipids. The current studies show that cells control their cholesterol content through receptor-ligand interactions and not through changes in the physical properties of the membrane.

A conformational switch controls the DNA cleavage activity of lambda integrase.

The bacteriophage lambda integrase protein (lambda Int) belongs to a family of tyrosine recombinases that catalyze DNA rearrangements. We have determined a crystal structure of lambda Int complexed with a cleaved DNA substrate through a covalent phosphotyrosine bond. In comparison to an earlier unliganded structure, we observe a drastic conformational change in DNA-bound lambda Int that brings Tyr342 into the active site for cleavage of the DNA in cis. A flexible linker connects the central and the catalytic domains, allowing the protein to encircle the DNA. Binding specificity is achieved through direct interactions with the DNA and indirect readout of the flexibility of the att site. The conformational switch that activates lambda Int for DNA cleavage exposes the C-terminal 8 residues for interactions with a neighboring Int molecule. The protein interactions mediated by lambda Int's C-terminal tail offer a mechanism for the allosteric control of cleavage activity in higher order lambda Int complexes.